Hydroxyproline Synthesis Research Articles

Hydroxylation of peptide-bound proline and lysine before and after chain completion of the polypeptide chains of procollagen

The synthesis of procollagen hydroxyproline and hydroxylysine was examined in matrix-free cells which were isolated from embryonic tendon by controlled enzymic digestion and then incubated in suspension. After the cells were labeled with [ 14C]proline for 2 min, or about one-third the synthesis time for a Pro-α chain, [ 14C]hydroxyproline was found in short peptides considerably smaller than the Pro-α chains of procollagen. The results, therefore, confirmed previous reports indicating that the hydroxylation of proline can begin on nascent chains. In similar experiments in which the cells were labeled with [ 14C]lysine, [ 14C]hydroxylysine was found in short, newly synthesized peptides, providing the first evidence that the hydroxylation of lysine can also begin on nascent peptides. However, further experiments demonstrated that the synthesis of hydroxyproline and hydroxylysine continues until some time after assembly of the polypeptide chains is completed.

Intracellular hydroxylation of non-helical protocollagen to form triple-helical procollagen and subsequent secretion of the molecule.

Cells liberated from the tendons of chick embryos by controlled enzymic digestion were used to examine the synthesis of the polypeptide chains of collagen separately from the secretion of collagen in the precursor form known as procollagen. When the cells were incubated under anaerobic conditions, the synthesis of collagen hydroxyproline was completely inhibited but the rate of protein synthesis remained about the same as the control rate for about 30 min. Under these conditions the cells synthesized the non‐hydroxylated precursor known as protocollagen and the protocollagen was retained in the cells. By incubating the cells with [14C]proline under anaerobic conditions and then exposing the system to air, it was possible to follow the intracellular hydroxylation of [14C]protocollagen to [14C]procollagen. The conformation of the intracellular [14C]protocollagen and [14C]procollagen was examined by proteolytic digestion under conditions in which the triple‐helical portion of collagen is resistant to digestion. When digestion with either pepsin or α‐chymotrypsin was carried out at either 30 ° C or 37 °C, all of the [14C]protocollagen was digested. After the same protein was hydroxylated to [14C]procollagen by exposing the cells to O2 a large fraction of the protein became resistant to proteolysis and was secreted. The results suggested that the intracellular hydroxylation of collagen polypeptides converts them from a non‐helical to a helical form and that the helical conformation probably is required for the protein to be secreted at an optimal rate.

Differing effects of 2,2-dipyridyl and oxygen on the synthesis of collagenous hydroxyproline in the cuticle and body wall of Ascaris lumbricoides

1. Adult specimens of Ascaris lumbricoides of similar weights were incubated under nitrogen for 24 hours in a synthetic medium with 1 mM 2,2′-dipyridyl. 2. Under these conditions, the viability of the parasites was not affected as evidenced by the amount of ATP in the whole sample and the mobility after mechanical stimulus. 3. Incorporation of [ 14C]proline into non-collagenous proteins in the body wall and cuticle was reproducibly higher in 2,2′-dipyridyl-treated specimens than in untreated worms. Synthesis of collagenous hydroxyproline was inhibited in the cuticle and, to a greater extent, in the muscle layer. 4. After transferring the specimens into a fresh medium enriched with 0·1 mM ferrous ions and incubated under 70% oxygen, the muscle collagen remained underhydroxylated. The synthesis of hydroxyproline, however, was almost completely normalized in the cuticle collagen. 5. We interpret the data as further evidence of the existence of at least two different enzymes hydroxylating collagenous proline, one located in the subcuticle and the other in the muscle layer of Ascaris lumbricoides.

Fatty acid inhibition of somatomedin (serum sulfation factor)-stimulated protein and RNA synthesis in embryonic chicken cartilage

The effects of fatty acids and serum containing somatomedin on in vitro synthesis of collagen, chondromucoprotein, general proteins and RNA were studied in embryonic chicken cartilage. Somatomedin (5 % rat serum added) stimulated 35SO 2− 4, [ 3H]leucine and [ 3H]tryptophan incorporation into chondromucoprotein-rich and collagen-rich cartilage protein fractions; and [ 3H]proline incorporation and [ 3H]hydroxyproline synthesis in the collagen-rich fraction. Somatomedin also stimulated [ 3H]uridine incorporation into RNA. In the non-stimulated state (no serum added) butyrate and octanoate had little or no effect on amino acid incorporation into protein or uridine incorporation into RNA. In the serum stimulated state, butyrate and octanoate inhibited amino acid incorporation into protein and uridine incorporation into RNA. Such inhibition was observed only with serum containing growth hormone-dependent sulfation factor (somatomedin). Agarose column chromatography of the chondromucoprotein-rich fraction revealed that while somatomedin stimulates synthesis of all proteins of this fraction equally, butyrate inhibits stimulated chondromucoprotein synthesis to a greater extent than certain other cartilage proteins (which are inhibited little, if at all). The data indicate that somatomedin stimulates embryonic chicken cartilage RNA, collagen, chondromucoprotein and general protein synthesis. Fatty acids have little or no effect on unstimulated cartilage macromolecular synthesis. The somatomedin stimulation of RNA, collagen and chondromucoprotein synthesis, and some, but not all of protein synthesis other than collagen or chondromucoprotein, is inhibited by fatty acids.

Total urinary hydroxyproline excreted in healing experimental rat rickets, studied with 14 C proline.

The healing of phosphate vitamin-D-deficient rachitic rats is accompanied by a two to sixfold increase in total urinary hydroxyproline excretion. In14C studies this increase is shown to be partly due to a higher rate ofde novo synthesis of hydroxyproline (OHPr) peptides in healing as compared with a control series of rachitic rats. There is no significant difference in the labelled carbon pool as shown by14CO2 excretion in the healing compared with rachitic rats. There is evidence for increased synthesis of non-extractable collagen from the healing rachitic rat epiphyseal tissue. Phosphate repletion is seen to stimulate and alter protein synthetic pathways in bone apart from the role it may play in mineralisation.

Hydroxyproline-rich cell wall protein (extensin): Biosynthesis and accumulation in growing pea epicotyls

Plant cell walls contain a glycoprotein component rich in the otherwise rare amino acid hydroxyproline. We examined the synthesis and accumulation of wall hydroxyproline during different states of elongation growth in pea epicotyls. Light-grown peas contained more wall hydroxyproline than their taller, dark-grown counterparts. When elongation was studied by marking growing stems in situ, there was a marked accumulation of wall hydroxyproline coincident with the cessation of elongation. Dividing and elongating regions of the epicotyl showed less wall hydroxyproline than did regions where elongation was no longer occurring. Hydroxyproline biosynthesis was examined by incubation of excised sections of tissues in various growth states in 14C-proline. The extent of conversion of these residues to 14C-hydroxyproline served as a measure of the rate of hydroxyproline synthesis. This rate was highest in tissues which had ceased elongation. The low rate of hydroxyproline synthesis in dividing and elongating cells was probably not due to the inability to hydroxylate peptidyl proline or to secrete proteins. These data show a positive correlation between the synthesis and accumulation of cell wall hydroxyproline and the cessation of cell elongation in pea epicotyls.

Hydroxyproline-rich cell wall protein (extensin): Role in the cessation of elongation in excised pea epicotyls

The synthesis and accumulation of cell wall hydroxyproline increases coincident with the cessation of elongation growth in pea epicotyls. We examined the relationship between these biochemical and physiological events by using epicotyl sections challenged with α, α′-dipyridyl. This chelator blocked hydroxyproline biosynthesis without affecting overall protein synthesis. Epicotyl sections mimicked elongation growth in situ when placed in indoleacetic acid. Elongation was blocked by the addition of benzimidazole or Ethrel. These latter compounds acted independently as judged by their kinetics of action and the inhibition of Ethrel's effect only by CO 2. During rapid elongation growth in indoleacetic acid, there was no increase in cell wall hydroxyproline. However, incubation in either growth-inhibitory agent increased hydroxyproline 3-fold. When this increase was blocked by dipyridyl incubation, growth was not inhibited in benzimidazole or Ethrel, but proceeded at the maximal rate. During long-term incubations in buffer, cell wall hydroxyproline increased and the sections eventually became unable to grow. However, if dipyridyl was added to block the hydroxyproline increase, growth potential remained. Elongation was inhibited by supraoptimal concentrations of indoleacetic acid. However, such inhibition did not occur in the presence of dipyridyl. These results indicate that an hydroxyproline-containing component is necessary in rendering the cell wall inextensible when elongation growth ceases.

Hydroxylation of (X-Pro-Gly) n by protocollagen proline hydroxylase Effect of chain length, helical conformation and amino acid sequence in the substrate

A series of peptides containing sequences of -X-Pro-Gly- were synthesized by a solid-state method which gave homogeneous products. These peptides were then used to study the synthesis of hydroxyproline by protocollagen proline hydroxylase. Hydroxylation of random-coil forms of the series of peptides (Pro Pro-Gly) n with n = 5, 10, 15 and 20 indicated no difference among the peptides in V but the K m decreased markedly with chain-length. Hydroxylation of (Pro-Pro Gly) 10 in the triple-helical conformation clearly demonstrated that the triple-helical conformation does not in itself prevent hydroxylation. The V for the helical form was 64% of the V for the random-coil form but the K m in molar concentrations was the same for both forms. Hydroxylation of the peptides Arg-Gly-(Pro-Pro-Gly) 5 and Glu-Gly (Pro-Pro Gly) 5 indicated that the presence of a basic amino acid in the NH 2-terminal end decreases the K m . Comparison of three more complex peptides indicated that modifications of amino acids in the -X-Pro-Gly- triplets which are hydroxylated as well as modified in adjacent triplets can affect the reaction with protocollagen proline hydroxylase.

Diverse effect of 1,10-phenanthroline and 2,2-′dipyridyl on the synthesis of collagen and noncollagenous proteins in in vivo and in vitro systems

Both ferrous iron chelators inhibit synthesis of collagenous hydroxyproline in the system of skin slices while the synthesis of noncollagenous proteins significantly stimulated by certain concentrations of these agents. When intact rats were injected with 1,10-phenanthroline for various times up to 14 weeks there was no effect on the amount or synthesis of hydroxyproline in the liver, while the synthesis of total noncollagenous liver proteins as well as microsomal proteins was increased following high doses of this agent. This finding is related to an inductive effect of this drug on the microsomal system while the in vitro effects are related to chelation.

Synthesis of Hydroxyproline in Corneal Epithelium

Hydroxyproline has been demonstrated in regenerating as well as in cultured corneal epithelial cells after exposure to tritiated proline. It seems, therefore, that the epithelium is capable of synthesizing at least precursors of collagen. Cycloheximide inhibited the synthesis of hydroxyproline. The question remains open whether the synthesis of hydroxyproline-containing peptides or proteins serves only to rebuild the epithelial basement membrane or contributes also to the reformation of stromal collagen.

INFLUENCE OF ALCOHOL ON COLLAGEN SYNTHESIS IN VITRO

The effect of a range of concentrations of ethanol on collagen biosynthesis in vitro was studied by measuring 14C-proline uptake and 14C-hydroxyproline production in embryonic chick tibia preparations. With increasing ethanol concentration there was decreasing proline incorporation. Hydroxyproline synthesis was relatively stimulated by low, but was progressively inhibited by higher, concentrations of ethanol. The effect of certain proprietary alcohols in this simple experimental preparation did not correlate with their ethanol content. The evidence indicates that certain of the congeners in alcoholic beverages are potentially cirrhogenic.

Stimulation of Collagen Synthesis in Embryonic Chick Skin by Insulin

The effect of insulin on collagen biosynthesis was studied using 14C proline and 14C lysine in 10 day embryonic chick skin. Insulin stimulated (in vitro) the total incorporation of both amino acids as well as the formation of 14C hydroxyproline and 14C hydroxylysine. The stimulatory effect of insulin was more pronounced with 14C proline when a 2-fold increase in 14C hydroxyproline synthesis was observed. When 14C lysine was present in the incubating medium, a 50% increment in 14C hydroxylysine formation was noted when insulin was added. Insulin had no effect on the glycosylation of newly formed hydroxylysine. Maximum stimulation by insulin occurred at 6 hours.

Asymmetry in the hydroxylation of (Pro-Pro-Gly) 5 by protocollagen proline hydroxylase

Peptides of the structure (Pro-Pro-Gly) 5 and labeled with [ 14C]proline in individual triplets were synthesized and used as substrates for the synthesis of hydroxyproline by protocollagen proline hydroxylase. The results indicated that the enzyme hydroxylated the peptide asymmetrically in that the fourth triplet from the NH 2-terminal end was hydroxylated to a greater extent than any other triplet.

Synthesis of hydroxyproline in the regenerating corneal epithelium

Synthesis of hydroxyproline in the regenerating corneal epithelium

Lag between incorporation of proline into protocollagen and synthesis of hydroxyproline during collagen biosynthesis in vitro and in vivo.

THE hydroxyproline and hydroxylysine in collagen are synthesized by hydroxylation of proline and lysine after these amino-acids have been incorporated into peptide linkages (for review see ref. 1). Experiments with embryonic cartilage in vitro in which the hydroxylases were intermittently inhibited demonstrated that the hydroxylations can occur after the proline-rich and lysine-rich polypeptide precursor protocollagen is released from ribosomal complexes1,2. There has been controversy, however, over the question of whether in uninhibited systems the hydroxylation of the appropriate prolyl and lysyl residues occurs while nascent polypeptide chains are still being assembled on ribosomes1,3,4.

Effect of calcitonin on (3H)proline incorporation into bone hydroxyproline in the rat.

SUMMARY The effect of calcitonin on the incorporation of [3H]proline into bone hydroxyproline was investigated in young growing rats. The action of the hormone was studied under the following conditions: (1) acutely, in both intact and thyroparathyroidectomized rats, (2) after 20 days of daily treatment in both intact and thyroparathyroidectomized animals, (3) in thyroparathyroidectomized animals treated with parathyroid extract for 20 days, and (4) in animals fed a low calcium diet for 25 days. Calcitonin had no effect when given either acutely or long-term to intact animals. [3H]Proline incorporation into bone hydroxyproline was increased in rats fed low calcium diet, in rats treated with parathyroid extract for 20 days, and in acutely thyroparathyroidectomized rats. In each instance, calcitonin prevented or reduced this increase. The results are interpreted as evidence that, with the doses used, hydroxyproline synthesis under normal conditions is unaffected by the hormone. However, where hydroxyproline formation was increased, calcitonin reduced this increase.

Hydorxyproline biosynthesis in plant cells Peptidyl proline hydroxylase from carrot disks

Hydroxyproline occurs in many plant proteins and is especially abundant in the proteins associated with the primary cell wall. In vivo evidence indicates that the synthesis of peptidyl hydroxyproline occurs by the hydroxylation of peptidyl proline. We have shown that plant cells contain an enzyme which catalyzes this hydroxylation reaction. The hydroxylase has the following characteristics: it requires O 2, Fe 2+, ascorbate and an α-keto acid for activity; it does not react with free proline; it is localized in the soluble cytoplasm; it readily hydroxylates prolyl residues of protocollagen isolated from chick embryos. A partial purification of the enzyme was achieved by the use of (NH 4) 2SO 4 and Ca 3(PO 4) 2 gel fractionations.

Environmental control of macromolecular synthesis in cartilage and bone: morphogenetic response to hyaluronidase.

Tibiae of 12-day embryonic chickens were grown in organ culture in synthetic medium for 48 h in the presence of various levels of hyaluronidase. This treatment: (1) removed between 49 and 86 % of hexosamine-containing material from the rudiments, and the level of hydroxyproline remained just below that in controls; (2) reduced the length and weight of the rudiments; (3) increased the total protein and DNA content in bone but reduced the amount gained in cartilage; (4) altered the fine-structure of the epiphysial chondroblasts; (5) failed to affect the rate of incorporation of isotopic uridine and leucine. When explants were subcultured in normal medium for a further 5-day period, the degree of recovery was dose-dependent; the final level of constituents in bone and cartilage of some rudiments exceeded those in controls. Hyaluronidase greatly enhanced the synthesis of hexosamine-containing materials during the period of treatment of the rudiments and a second, but generally smaller, wave of excess synthesis occurred during the recovery phase; initially, large amounts of polymer chains were released into the medium, but they were incorporated into the tissues rapidly towards the end of culture. Characterization of the glycosaminoglycans indicated, also, that an abnormal type of polymer chain was synthesized and rejected by the tissue. The rate of synthesis of both hydroxyproline, and hence collagen, and non-collageneous protein was also enhanced during the recovery period. The results showed that the cells could re-establish their normal environment following the initial loss of intercellular macromolecules, and that such changes in the concentration of constituents enhanced the synthetic responses of the cells.

Effect of increased oxygen of hydroxyproline synthesis in the cuticle and body wall of Ascaris lumbricoides

1. 1. Ascaris lumbricoides injected with [ 14C]proline were incubated for 24 h in 70% O 2 at normal pressure and in air at 1.6 atm abs. 2. 2. In Ascaris incubated at high O 2 concentration a substantial increase in collagenous proline hydroxylation occured (1.6–3 times) in the cuticle while little effect of perhaps a decrease in hydroxyproline synthesis was observed in the body wall. 3. 3. When incubating Ascaris at four varying concentrations of O 2 (0, 5, 20 and 70%) the hydroxylation of proline (given as ration specific activity hydroxyproline/specific activity proline) measured in purified cuticle collagen was maximal when the worms were under 70% O 2 and 4 times higher than when under N 2. In the case of muscle collagen, hydroxylation was maximal at 20% O 2; 70% O 2 was no better than N 2. 4. 4. The results are discussed in terms of two different protocollagen proline hydroxylases; one in the cuticle stimulated by O 2 and one in the body wall that is perhaps slightly inhibited by high O 2. Alternatively, the results may be due to differences mainly in the availability of O 2 to protocollagen hydroxylase in the two body layers. 5. 5. Data are presented on the distribution of various forms of collagenous hydroxyproline in Ascaris cuticle and body wall.

A method for studying collagen biosynthesis in human skin biopsies in vitro

Biosynthesis of collagen was studied by incubating slices of human skin in a medium containing [ 14C]proline. The presence of hydroxy[ 14C]proline in nondialysable protein of the skin was taken as a measure of collagen formation. The synthesis of radioactive hydroxyproline was studied under various experimental conditions, and an incubation medium (20 mM N-2-hydroxyethylpiperazine- N′-2-ethanesulphonic acid buffer (pH 7.4), so mM glucose, 0.05 mg/ml ampicillin, I μC [ 14C]proline, all in phosphate-free Krebs-Ringer solution) for an optimal rate of collagen synthesis is presented. The separate components of the incubation medium had no inhibitory effect on the hydroxylation of [ 14C]proline when partially purified preparations of protocollagen proline hydroxylase were incubated with radioactive protocollagen substrate. When skin biopsy specimens from living human subjects of various ages were used for incubation, younger subjects demonstrated a relatively high rate of collagen synthesis and a decrease with advancing age was observed.